Western Blotting Reagents Roundup – July 2022

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Western Blotting

Azure Biosystems supplies reagents for every step of the Western blotting workflow, from transfer to blocking to detection. Keep reading for brief summaries of four publications in which researchers used Azure Biosystems reagents for Western blotting and Western blot quantitation in their studies.

Phosphorylated MED1 links transcription recycling and cancer growth

Aberrant transcription goes hand-in-hand with oncogenesis. Chen et al1 used Azure’s Chemi Blot Blocking Buffer and Radiance ECL in Western blot experiments investigating transcription recycling in cancer cells. Uncontrolled transcription initiation and elongation are known to be associated with tumor growth but the authors examined whether Pol II recycling, in which RNA polymerase II re-transcribes the same gene rather than being released after transcription is complete, is also associated with cancer. Using a recycling assay developed in their prior publications, the authors demonstrated that Mediator 1 (MED1), when phosphorylated by CDK9, drives Pol II recycling.

Phosphorylation of MED1 increased during prostate cancer progression and inhibiting CDK9 decreased MED1 phosphorylation, Pol II recycling, and prostate tumor growth. The results suggest MED1 phosphorylation and transcription recycling are involved in cancer growth, and MED1 phosphorylation may provide a biomarker to assess therapeutic response of cancers to CDK9 inhibitors.

DiscoverChemi Blot Blocking BufferRadiance ECL

Aripiprazole Offsets Mutant ATXN3-Induced Motor Dysfunction by Targeting Dopamine D2 and Serotonin 1A and 2A Receptors in C. elegans

Machado-Joseph disease (MJD) is a dominantly inherited progressive ataxia caused by expansion of a CAG repeat in the ataxin-3 gene. Jalles et al2 used Azure’s Radiance ECL and AzureRed total protein stain, in addition to the Sapphire Biomolecular Imager, in a study investigating how the antipsychotic drug aripiprazole suppresses MJD pathogenesis. In a C elegans model of MJD, the authors found that aripiprazole improved motor performance and this improvement depended on dopamine D2-like and serotonin 5-HT1A and 5-HT2A receptors. Identifying the specific targets of aripiprazole may help develop new therapeutics for MJD with fewer side effects.

Discover: Radiance ECLAzureRedSapphire Biomolecular Imager

MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization

During metastasis, cancer cells migrate by building out the cytoskeleton at the leading edge of the cell and retracting it at the rear. Pasapera et al3 used Azure’s Radiance ECL in a study of cancer cell cytoskeleton polarization. The authors investigated whether the kinase MARK2, known to regulate the microtubule cytoskeleton in other processes, plays a role in the polarization of the cytoskeleton and directed migration of cancer cells. In osteosarcoma cells, Western blot experiments demonstrated that MARK2 promotes stress fiber formation and myosin II activation and mediates inactivation of myosin phosphatase.

The data suggests MARK2 is a major regulator of cell contractility and adhesion that mediates cancer cell motility.

Discover: Radiance ECL

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2β1

Alport syndrome is a congenital, progressive glomerular disease that leads to the progressive loss of kidney function. Madison et alused Azure’s Radiance ECL and TotalStain Q as well as the Azure 600 Imaging System in a study characterizing the glomerular basement membrane (GBM) in a mouse model of Alport syndrome. The investigators found that collagen a1(III) was deposited in the GBM of Alport mice; in wild type mice, collagen a1(III) is found only in the mesangium.

Quantitative Western blotting was carried out using total protein normalization with TotalStain Q staining as the control and the quantitative Westerns confirmed increased levels of collagen a1(III) in the glomeruli of Alport mice. The presence of collagen a1(III) was found to activate DDR1 receptors and lead to changes in gene expression consistent with podocyte injury. Lack of either of the two collagen receptors on podocytes has previously been shown to slow disease progression. The results indicate aberrant collagen-mediated co-receptor signaling through the DDR1 and a2b1 integrin receptors contribute to podocyte injury and renal pathology in Alport syndrome.

Discover: Azure 600 Imaging SystemRadiance ECLTotalStain Q

Find more publications using Azure reagents and imaging systems on the Azure publications list, or contact us directly for assistance with a specific product by using the form on the left.

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SOURCES

  1. Chen Z, Ye Z, Soccio RE, et al. Phosphorylated MED1 links transcription recycling and cancer growth. Nuc Acids Res. 2022;500(8):4450-4463.
  2. Jalles A, Vieira C, Pereira-Sousa J, et al. Aripiprazole offsets mutant ATXN3-induced motor dysfunction by targeting dopamine D2 and serotonin 1A and 2A receptors in elegans. Biomedicines. 2022;10(2):370.
  3. Pasapera AM, Heissler SM, Eto M, et al. MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization. Curr Biol. 2022;32(12):2704-2718.
  4. Madison J, Wilhelm K, Meehan DT, et al. Glomerular basement membrane deposition of collagen a1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin a2b J Pathol. 2022; doi: 10.1002/path.5969.

Intranasal Administration of Neutralizing IgA Increases SARS-CoV-2 Infection in a Hamster Model

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COVID-19 Western Blotting

Infectivity of SARS-CoV-2 is associated with high viral loads in the upper respiratory tract. Alpha and gamma immunoglobulins (IgA and IgG) both neutralize viral infections, with IgA significant in mucosal immunity and IgG predominantly affecting blood. Most studies focus on IgG in SARS-CoV-2 infection, but the role of IgA is less understood.

To enter host cells, the receptor binding domain (RBD) of the SARS-CoV-2 spike protein binds to cellular ACE2 receptors. The spike protein is cleaved and the viral and cellular membranes fuse, allowing the virus into the cell. The spike protein is the target of current SARS-CoV-2 vaccines and is the major antigen target of antibodies in infected individuals. Additionally, RBD-specific IgA have been found in COVID-19 patients.

In a preprint shared on ResearchSquare, Zhou et al investigated whether intranasal IgA could protect against SARS-CoV-2 infection in Syrian hamsters. The study is currently under review so the current version is not peer-reviewed or published in a journal. The authors report that neutralizing IgA antibodies, applied intranasally before SARS-CoV-2 challenge, reduced the amount of virus in the lungs but increased viral infectivity in the nasal turbinate of the hamsters.

The authors isolated and characterized 18 RBD-specific monoclonal antibodies from four COVID-19 patients. Five of these showed neutralizing activity against SARS-CoV-2. The one with the tightest binding to RBD was tested in the hamster model to see if intraperitoneal administration of the antibody before or soon after viral challenge would affect infection. It did decrease viral loads in the lungs but did not prevent infection in the nasal turbinate.

The group then engineered monomeric and dimeric IgA1 and IgA2 versions of the antibody and tested these in hamsters, either by intraperitoneal injection or nasal administration. The monomeric IgA1 but not IgA2 protected lungs, while neither prevented nasal infection. When dimeric versions of the IgA1 and IgA2 were tested, they were found to decrease infection in the lungs but increase infection and enhance damage in the nasal turbinate.

Why would dimeric IgA enhance SARS-CoV-2 infection? The authors found that the IgA-mediated enhancement did not depend on the ACE2 receptor. Instead, the targets were dendritic cells expressing CD209, a lectin known to be a receptor for secretory IgA.

In the course of this work, the authors used the Sapphire Biomolecular Imager in viral neutralization assays and assays of enhanced infection via CD209. For each assay type, cells were grown in 96-well plates and infected with SARS-CoV-2. The media was removed, cells permeabilized and SARS-CoV-2 was detected with an rabbit anti-virus primary antibody followed by a goat anti-rabbit secondary antibody labeled with Alexa Fluor 488. The resulting fluorescent signal was detected on the Sapphire.

In addition to fluorescence imaging, the Sapphire Biomolecular Imager provides densitometry, phosphor, multichannel fluorescence, chemiluminescence and white light imaging of blots, gels, tissues, and more.

 

Explore: Azure Sapphire Biomolecular Imager

Western Blotting Reagents Roundup – September 2020

Categories
Multiplex Western Blotting

Azure offers products to support every step of your Western blotting workflow, from blocking to detection. Several recent publications highlight the use of Azure reagents in chemiluminescent and fluorescent Western blotting, in a variety of experimental systems and approaches. Below is a brief summary of four articles in which researchers incorporated Azure’s protein-free blocking buffer, secondary antibody conjugates, and ECL detection reagents into their research.

Adipocyte REVERBα dictates adipose tissue expansion during obesity

In a recent pre-print, Hunter et al recently demonstrated that REVERBa, a circadian clock component, modulates white adipose tissue metabolism in response to changes in metabolic state. The researchers created a mouse model in which REVERBa was knocked out in only white adipose tissue and compared these mice to mice missing REVERBa in all tissues. Chemiluminescent and fluorescent Western blots were used to confirm protein changes in knock out mice, and to follow circadian changes in protein levels. All Western blot experiments used Azure’s Protein-Free Blocking Buffer to block nitrocellulose membranes. This blocking buffer is a great choice to enhance signal and reduce background for general Western blots and is compatible with all detection chemistries.

Molecular Targeting of Cancer-Associated PCNA Interactions in Pancreatic Ductal Adenocarcinoma Using a Cell-Penetrating Peptide

Smith et al identified a novel potential therapeutic approach for pancreatic cancer. In Molecular Therapy Oncolytics, the researchers examined whether a peptide that mimics a part of the proliferating cell nuclear antigen (PCNA) could interfere with PCNA-protein interactions in pancreatic cancer cells. They found that the peptide killed cancer cells by inhibiting DNA replication and DNA repair, resulting in the accumulation of DNA damage. Fluorescent Western blots were used to assess DNA damage by detecting phosphorylated histone H2AX, a biomarker for double stranded DNA breaks. The secondary antibody used was the Azure Spectra 800 Secondary Fluorescent Antibody.

Poxvirus-encoded decapping enzymes promote selective translation of viral mRNAs

Cantu et al identified a surprising new function of vaccinia virus decapping enzymes. The decapping enzymes D9 and D10 are known to promote degradation of viral and cellular mRNAs. In PLOS Pathogens, the researchers compared genome-wide translation efficiency in cells infected with wild type vaccinia virus vs mutant virus lacking functional decapping enzymes. The results indicated that paradoxically, while promoting degradation of mRNAs, the decapping enzymes are also required for selective translation of certain post-replicative viral proteins whose mRNAs have 5’-poly(A) leaders. Translated proteins were detected on chemiluminescent western blots using Azure HRP-conjugated Secondary Antibodies.

2‐Oxothiazolidine‐4‐carboxylic acid inhibits vascular calcification via induction of glutathione synthesis

Patel et al are investigating the potential of a cysteine prodrug, 2‐oxothiazolidine‐4‐carboxylic acid (OTC) to inhibit arterial medial calcification. In a recent article published in the Journal of Cell Physiology, the researchers used Azure’s Radiance ECL HRP substrate to detect markers of cell differentiation in cultured vascular smooth muscle cells (VSMC), grown in a calcifying medium with or without OTC. Western blotting revealed that treatment with OTC reversed the increase in osteoblast markers and rescued the drop in glutathione synthesis enzymes observed in calcifying cells, leading the authors to conclude the drug was effective in vitro and might have clinical relevance. The long-lasting signal and high sensitivity of Radiance ECL substrate allows for reliable comparisons of protein levels between samples.

You can find more publications using Azure reagents and imaging systems on the Azure publications list, or contact Azure for assistance identifying publications using a specific product.

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